The Two Giant Sister Species of the Southern Ocean, Dissostichus

Polar Biol DOI 10.1007/s00300-006-0222-6

ORIGINAL PAPER

The two giant sister species of the Southern Ocean, Dissostichus eleginoides and Dissostichus mawsoni, diVer in karyotype and chromosomal pattern of ribosomal RNA genes

L. Ghigliotti · F. Mazzei · C. Ozouf-Costaz · C. Bonillo · R. Williams · C.-H. C. Cheng · E. Pisano

Received: 5 July 2006 / Revised: 27 September 2006 / Accepted: 28 September 2006 © Springer-Verlag 2006

Abstract The two giant notothenioid species, the ping to its additional submetacentric chromosome. The Patagonian toothWsh Dissostichus eleginoides and the additional rRNA genes in D. mawsoni may be a cold- Antarctic toothWsh D. mawsoni, are important com- adaptive compensatory mechanism for growth and ponents of the Antarctic ichthyofauna and heavily development of this large species in freezing seawater. exploited commercially. They have similar appearance and size, both are piscivorous and bentho- Keywords Antarctica · Chromosomes · Dissostichus · pelagic, but diVer in their geographic distribution and Ribosomal genes · ToothWsh absence/presence of the antifreeze trait. We karyo- typed these two sister species by analyzing specimens collected from multiple Antarctic and sub-Antarctic Introduction sites. Both species have a diploid number of 48, but diVer in karyotypic formula, (2m + 2sm + 44a) for D. The two species of toothWsh, the Patagonian toothWsh, eleginoides and (2m + 4sm + 42a) for D. mawsoni, Dissostichus eleginoides Smitt 1898, and the Antarctic due to an extra pair of submetacentric chromosomes toothWsh, D. mawsoni Norman 1937, are important in the latter. Chromosomal Xuorescence in situ components of the Southern Ocean ichthyofauna (Gon hybridization with rDNA probes revealed unex- and Heemstra 1990). They are similar in appearance, pected species-speciWc organization of rRNA genes; body size (»100 kg), piscivory and bentho-pelagic life D. mawsoni possesses two rDNA loci (versus one style (Eastman and DeVries 2000; Horn 2002). Both locus in D. eleginoides), with the second locus map- species are usually caught between 200 and 2,000 m depth (Hanchet et al. 2003), but diVer in geographic distribution. D. eleginoides is largely subantarctic and L. Ghigliotti · F. Mazzei · E. Pisano (&) occurs in the Ross Sea, along the Antarctic Peninsula, Department of Biology, University of Genova, around subantarctic islands in the Atlantic and Indian Viale Benedetto XV 5, 16132 Genova, Italy sectors of the Southern Ocean, and on the continental e-mail: [email protected] shelf oV the coasts of Uruguay, Argentina and Chile C. Ozouf-Costaz · C. Bonillo (Brickle et al. 2005). Conversely, D. mawsoni occurs Département Systématique et Evolution, around mainland Antarctica, generally south of 60°S CNRS UMR 7138, Museum National d’Histoire Naturelle, (DeWitt et al. 1990). The distribution of the two spe- 43 rue Cuvier, 75005 Paris, France cies overlaps north of the Ross Sea, in CCAMLR R. Williams (Commission for the Conservation of Antarctic Marine Antarctic Division, Channel Highway, Living Resources) Subarea 88.1 (Horn 2002) and on Kingston, TAS 7050, Australia BANZARE Bank, i.e. the southern part of the Ker- guelen Plateau in CCAMLR Division 58.4.3b C.-H. C. Cheng Department of Animal Biology, University of Illinois, (CCAMLR 2006). Confusion over species identity in 515 Morrill Hall, Urbana, IL 61801, USA some areas has sometimes been noticed, mainly oV the 123 Polar Biol

South Orkney/Antarctic Peninsula and the Southern the Weddell Sea (Ozouf-Costaz et al. 1991). The iden- Kerguelen Plateau (Fischer and Hureau 1985; Gon and tiWcation of the homologous chromosome pairs in this Heemstra 1990). specimen was diYcult due to the poor quality of the The two Dissostichus species are the major Wsh sample, but the diploid number and karyotypic for- resource exploited in the Southern Ocean by interna- mula could be determined and they are identical to tional longline and trawl Wsheries, with signiWcant those of the D. eleginoides (Ozouf-Costaz et al. 1991). international management eVorts under CCAMLR In the present work we deWne the speciWc chromo- jurisdiction (Eastman and DeVries 2000; Hanchet some set of each of the two toothWsh species by using et al. 2003; Duhamel et al. 2005). Recent escalation in conventional karyotyping and molecular cytogenetics commercial Wshing ventures, directed speciWcally and provide for the Wrst time an accurate description of towards the Antarctic toothWsh, has increased concerns the basic chromosomal features of these two important over the sustainability of the D. mawsoni Wshery, due Wsh species. This was accomplished by examining the to the scant ecological and life history information chromosome set of several specimens of both sexes, available on this species (Parker et al. 2002; Hanchet collected from multiple Antarctic and sub-Antarctic et al. 2003). locations. A more in-depth characterization was Physiologically, the two species diVer notably in their obtained through cytogenetic mapping of ribosomal capability to live at subzero temperatures. D. mawsoni genes by Xuorescence in situ hybridization (FISH), has a large family of antifreeze glycoprotein (AFGP) which revealed unexpected species-speciWc patterns in genes and produces high levels of circulatory AFGPs these two sister taxa. that prevent the freezing of its body Xuids (Chen et al. In the broader context of comparative genomics, the 1997; DeVries and Cheng 2005). In contrast, D. elegino- cytogenetic data presented here provide the necessary ides has barely detectable AFGP sequences in its DNA, basis for approaching further analyses of the genomes and no antifreeze activity in its blood (Cheng and of these two important species through the visualiza- Detrich 2006). This distinct diVerence is consistent with tion of gene loci at the chromosomal level. The use of the separate geographic distribution of the two tooth- speciWc DNA or gene sequences as probes in chromo- Wsh in freezing versus non-freezing habitats. The evolu- somal FISH is a powerful tool that provides spatial tionary history of these two species in disparate thermal landmarks and linkage information in a genome that environments suggests other signiWcant molecular and can help resolve positional ambiguities in whole biological diVerences are likely to exist, despite their genome sequence assemblies, as well as facilitate the close phylogenetic kinship. elucidation of gene family and genome organizations Taxonomically, the genus Dissostichus belongs to and evolution (Cheung et al. 2001; Jaillon et al. 2004). Nototheniidae, the most diversiWed family of the suborder Notothenioidei, and it is presently included in the subfamily Pleuragrammatinae (Balushkin 2000) Materials and methods together with the other pelagic genera Pleuragramma, Aethotaxis and Gvozdarus. According to phylogenetic Animal sampling and chromosome preparation analyses, Dissostichus is a basal nototheniid lineage. Using penalized likelihood analysis combined with fos- Specimens of D. eleginoides were collected in the sil calibration, the age of most recent common ancestor Indian and Atlantic sectors of the Southern Ocean. of the two Dissostichus species was estimated to be The specimens from the Indian sector (four females, about 14.5 My (Near 2004). three males and one undetermined juvenile) were Despite substantial scientiWc attention to the tooth- caught by use of bottom trawls during the “Thirst” Wsh related to biodiversity conservation, Wshery man- Cruise 1993 aboard the Australian R/V Aurora Aus- agement (Parker et al. 2002; Hanchet et al. 2003) and tralis (around 51°/52°S, 72°/75°E). The specimens from molecular analyses of the antifreeze trait (Chen et al. the Atlantic sector (six females) were collected by 1997; Cheng and Detrich 2006), cytogenetic analyses of traps during the “ICEFISH” Cruise 2004 aboard the the two species are presently scanty. On the basis of a United States R/V Nathaniel B. Palmer (at 54°S, 55°W single juvenile caught oV the Kerguelen Islands, Dous- approximately). sau De Bazignan and Ozouf-Costaz (1985) provided Specimens of D. mawsoni, two males and two preliminary data on the diploid number (48) and females, were collected in McMurdo Sound (78°S, karyotypic formula (44a + 2sm + 2m) of D. eleginoides. 167°E) by winched cable and hook through large holes For D. mawsoni, the only available karyotypic infor- drilled through sea ice. Other specimens were caught mation is based on the analysis of a single female from by bottom trawls during the Western Ross Sea Voyage 123 Polar Biol

2004 aboard the New Zealand R/V Tangaroa, one Wcation of genomic DNA. On the basis of Notothenia female in the Ross Sea (at 72°S, 173°E approximately) coriiceps 28S ribosomal DNA partial sequences (Gen- and two females around the Balleny Islands (67°S, Bank accession numbers AY141673 and AY141674), a 164°E). Six specimens (females) were caught by gill pair of primers, Noto_28S_C1 For (5Ј-CTA ACC nets at the French Station Dumont D’Urville (Adélie AGG ATT CCC TCA GTA GCG G-3Ј) and Land) (64°S, 140°E). Noto_28S_D3 Rev (5Ј-CAG GCA TAG TTC ACC Specimens were maintained in tanks or aquaria sup- ATC TTT CGG GTC-3Ј) were designed for ampliWca- plied with fresh, aerated seawater at local ambient tion. The PCR product was cloned in the pGEM-T temperature aboard the scientiWc vessels or at the Ant- Easy vector (Promega) and sequenced with BigDye v.3 arctic science stations. Specimens were injected with chemistry (Applied Biosystems). The sequence of colchicine and mitotic cells were obtained from head clone 28S_C1D3 has been deposited under GenBank kidney and spleen, following the method of Doussau accession number AY926497. The 28S_C1D3 clone de Bazignan and Ozouf-Costaz (1985) with slight mod- was used as a probe for FISH after nick translation iWcations. Fixed cells were stored at ¡20°C for further labeling with biotin¡16-dUTP (Roche Diagnostics) analyses. according to standard procedures.

Karyotyping 5S rDNA probe

Chromosome spreads on microscope slides were The DNA fragment used as probe for locating the 5S treated for conventional karyotyping and chromosome ribosomal genes is a partial sequence (86 bp) from the banding according to current protocols for nototheni- highly conserved 5S rRNA coding region. It was oid Wshes (Ozouf-Costaz et al. 1997). Characterization obtained by PCR ampliWcation of D. mawsoni genomic of chromosomal morphology followed the standard DNA using the primers: CB-5S-R1 For (5Ј-CAC CTG nomenclature according to centromeric position and GTA TTC CCA GGC-3Ј) and CB-5S-F2 Rev (5Ј-TAC arm ratio (Levan et al. 1964; Klinkhardt et al. 1995), as GCC CGA TCT CGT CCG ATC-3Ј). The PCR prod- metacentric (m), submetacentric (sm), subtelocentric uct was sequenced in an automated DNA sequencer (st) and acrocentric (a). Metacentric and submetacen- (Applied Biosystems). The sequence has been depos- tric chromosomes are bi-armed, whereas the acrocen- ited under GenBank accession number DQ478734. tric ones are one-armed. In the bi-armed elements the The probe was labeled with digoxigenin-11-dUTP long arm is referred to as q (queue) and the short as p (Roche Diagnostics) by nick translation according to (petit), according to the International system for standard procedures. human cytogenetic nomenclature (Mitelman 1995). The total number of chromosome arms in a karyotype Fluorescence in situ hybridization constitutes the fundamental number (FN). The chromosomes were arranged in the karyotypes according to The labeled probes were puriWed by ethanol precipita- morphology and size. tion and dissolved individually or together in the The karyotypes of the two Dissostichus species were hybridization buVer (50% formamide/2 £ SSC, 40 mM W established by examination of specimens collected KH2PO4, 10% dextran sulfate) to yield nal concentra- from each of the diVerent geographic areas. Multiple tions of 10 ng/l (28S rDNA) and 20 ng/l (5S rDNA). DAPI-stained metaphases from each specimen were One-color FISH and two-color FISH were per- analyzed with an Olympus BX61 equipped with a Sen- formed as described previously (Mazzei et al. 2004). sys (Photometrics) CCD camera for digital imaging. BrieXy, the chromosomes were denatured by heating Micrographs were processed either by the use of at 70°C for 1 min in 70% (v/v) formamide/2 £ SSC (pH Genus Software (Applied Imaging) or by application 7), dehydrated in a cold ethanol series, and air-dried. of Adobe Photoshop and Corel Photopaint image anal- The probes were applied to chromosomal spreads ysis software. (20 l per slide) and incubated overnight in a moist chamber at 37°C. High-stringency post-hybridization Ribosomal DNA probes washing was performed in 2 £ SSC at 72°C (5 min) followed by 2 min in PBD buVer (4 £ SSC, 0.07% 28S rDNA probe Tween20) at room temperature. Hybridized 28S rDNA probe was detected by incubation of chromosomal A partial sequence (762 bp) from the major ribosomal spreads with streptavidin-FITC (MP Biomedicals), and RNA genes of D. mawsoni was derived by PCR ampli- 5S rDNA probe by rhodamine-anti-digoxigenin antibody 123 Polar Biol

(Roche Diagnostics). Fluorescence signals of two-color morphology in terms of length variability of the p FISH were visualized by simultaneous co-application (short) arms. This size polymorphism of the homo- of the two detection reagents. The chromosomes were logues of pair 2 does not occur in all specimens, such counterstained in 0.3 g/ml DAPI/2 £ SSC and mounted that homomorphic and heteromorphic combinations in a standard anti-fade solution (Vector). are observed in specimens regardless of their geo- Chromosomal spreads were analyzed using a Zeiss graphic location. All the other elements (44) of the Axiophot Xuorescence microscope, and Xuorescence karyotype are acrocentric chromosomes in decreasing signals were captured by use of a cooled CCD camera size (Fig. 1a). The karyotypic formula for D. elegino- and processed with the software Genus for animal ides is 2m + 2sm + 44a. No sex-related karyotypic chromosomes (Applied Imaging). diVerences were observed. Similar to D. eleginoides, all specimens of D. mawsoni presented 48 chromosomes, arranged in 24 pairs Results as shown in the karyotype (Fig. 1b). However, the FN (54) and the karyotypic formula (2m + 4sm + 42a) are The karyotypes diVerent, due to the presence of an additional pair of submetacentric chromosomes, and 42 instead of 44 Examination of multiple metaphase plates from the acrocentric chromosomes. The metacentric elements of various specimens indicated that the two toothWsh have pair 1 are the smallest of the complement and likely a diploid number of 48 but diVer in their FN, 52 for D. corresponding to chromosome pair 1 of D. eleginoides. eleginoides versus 54 for D. mawsoni. The submetacentric chromosomes comprising pair 2 The karyotype of D. eleginoides is shown in Fig. 1a. appear very similar in morphology to the correspond- Among the 24 chromosome pairs comprising the set, ing chromosomes of pair 2 in D. eleginoides, including pair 1 is assigned to the smallest elements, which are frequent heteromorphism of the p arm size. The addi- metacentric and easily recognizable. Chromosome pair tional pair of submetacentric chromosomes in D. maw- 2 is also recognizable in all the metaphase spreads as soni (pair 3) does not correspond morphologically to being the only pair of submetacentric chromosomes. any element in the karyotype of D. eleginoides. Simi- Pair 2 shows a certain degree of heteromorphism in larly to pair 2, the two submetacentric homologs

Fig. 1 The karyotypes of the Patagonian toothWsh Dissostichus eleginoides (a) and Antarctic toothWsh D. mawsoni (b). DAPI staining. Bar =10m

123 Polar Biol comprising pair 3 in D. mawsoni are usually hetero- or homomorphism of the arm length. The 5S ribosomal morphic due to diVerences in size between their p arms. probe hybridizes to the same chromosome pairs and No sex-related karyotypic diVerences were observed. arm locations, indicating that the 5S ribosomal genes closely cluster with the major rRNA genes (Fig. 2b). Chromosomal location of the ribosomal RNA genes The FISH results for D. mawsoni unambiguously show that two pairs of chromosomes (pairs 2 and 3), In D. eleginoides the hybridization signal of the major instead of one, carry clusters of major ribosomal genes ribosomal RNA gene probe 28S_C1D3 was localized (Fig. 2c). In both pairs, the clusters of genes extend on chromosome pair 2 (Fig. 2a). In all the specimens along the whole length of the chromosome p arms the clusters of major ribosomal genes occupy the entire regardless of the heteromorphic length variations p arms of the homologs regardless of heteromorphism between specimens. The 5S ribosomal genes colocalized

Fig. 2 Metaphase chromosomes from Dissostichus eleginoides (a, b) and D. mawsoni (c, d) after Xuorescence in situ hybridization to map the location of major ribosomal genes (green signals in a, c) and 5S ribosomal genes (red signals in b, d). Numbers in c and d indicate the rDNAs bearing chromosomes of pairs 2 and 3 in D. mawsoni. Bars =10m

Fig. 3 Details of rDNAs bearing chromosome pairs, from diVer- (b, f); FISH with the 28S rDNA probe (c, g); FISH with the 5S ent metaphases in D. eleginoides (a–d), and D. mawsoni (e–h) af- rDNA probe (d, h) ter banding and FISH. DAPI banding (a, e); Ag-NOR banding 123 Polar Biol with the major rDNA clusters on the short (p) arms of pair 2 are always recognizable in the metaphase plates the submetacentric chromosome pairs 2 and 3 (Fig. 2d). by their similar shape, size and structure, supporting The two rDNA-bearing chromosomal regions in D. their common origin. The distinct diVerence between eleginoides and the four in D. mawsoni share the com- the karyotypes of the two Dissostichus species is the mon features of weak DAPI staining intensity (Fig. 3a, presence of an additional pair of bi-armed elements in e), and comprise the Nucleolar Organizing Regions D. mawsoni (chromosome pair 3) that has no morpho- (NORs), as revealed by chromosomal banding with sil- logical equivalent in the karyotype of D. eleginoides, ver nitrate (Fig. 3b, f). Since the Ag-based techniques leading to a diVerent karyotypic formula. In addition, detect NORs by staining a complex of acidic protein the FISH analysis demonstrated that ribosomal DNA associated with the Wbrillar center of the nucleolus and occurs along the whole length of the p arms of chromo- nascent pre-RNA (Jordan 1987), the positivity to silver some pair 3, thus revealing a fundamental genomic/ nitrate treatment indicates that ribosomal genes from karyotypic diVerence between the two species: in D. all the chromosomal locations have been actively tran- eleginoides the multigenic units of rDNA are organized scribed during the cell cycle. in a single locus on chromosome pair 2, whereas in D. mawsoni rDNA sequences are clustered in two separate loci on two separate chromosome pairs, namely Discussion pairs 2 and 3 (Figs. 2, 3). The rDNA chromosomal loci, regardless of their The toothWsh karyotypes number, share characteristics that indicate a common chromosomal organization. The nucleolar ribosomal Given the recognized monophyly of the Notothenioi- genes, detected with the 28S rDNA probe, are located dei suborder (Near et al. 2004), cytogenetic evidence along the entire p heterochromatic arm, which also supports the hypothesis that the putative ancestor of comprises the nucleolar organizer regions (NORs) as the Notothenioidei had a karyotype close to that of the detected by AgNO3 staining (Fig. 3). The extranucleo- basal lineage Bovichtidae, consisting of 48 one-armed lar rDNA 5S repeats, detected with the 5S rDNA (acrocentric) chromosomes (Mazzei et al. 2006). By probe, colocalize with the nucleolar ribosomal genes. this reasoning, the karyotypes of the extant species of Both D. eleginoides and D. mawsoni show size hetero- the more derived lineages should have arisen from morphism in the homologous chromosome bearing rearrangements of the presumed ancestral set of 48 rDNA genes, indicating that various amounts of the acrocentric chromosomes. The basal phylogenetic ribosomal repeats can be present per haploid comple- position of Dissostichus among Antarctic nototheni- ment. The heteromorphic length variation of the chro- oids (Near 2004) is consistent with our observed karyo- mosome regions bearing rDNA genes of the two types of 48 chromosomes, the majority of which Dissostichus species is a common organization found in consists of acrocentric elements resembling the puta- all Antarctic notothenioids (Mazzei et al. 2004). The tive basal set of 48 one-armed chromosomes. The pres- linked conWguration of the 28S-5S ribosomal genes is ence of two-armed chromosomes (pairs 1 and 2) in the also a common feature in cold-adapted notothenioids karyotypes of the two Dissostichus species represents a (Mazzei et al. 2006). derived karyotypic feature that could have arisen in Among Wshes in general, chromosomal organization their common ancestor by pericentric inversions in a of rRNA genes is highly variable. The 28S and 5S pair of acrocentric chromosomes. Pericentric inver- rRNA genes can occur separately or linked (reviewed sions lead to changes in chromosome formula but not in Martins and Galetti 2001; Martins 2006), and in sin- in diploid number, and are among the most common gle or multiple chromosomal locations (Sola et al. modiWcations contributing to karyotypic rearrange- 2000; Almeida-Toledo et al. 2002) even within the ment in Wsh, as well as in other vertebrates (White same species (Almeida-Toledo et al. 2002; Boron et al. 1973; King 1993). This type of rearrangement, together 2006). According to present information, a single chro- with Robertsonian rearrangements (fusion or Wssion of mosomal location of linked 5S and 28S rDNAs is the chromosomes) and reciprocal translocations, have predominant condition among Antarctic notothenioids been recognized as responsible for some of the impor- (Pisano and Ozouf-Costaz 2003; Mazzei et al. 2004). tant changes leading to the present karyotypic diversity The dual rDNA loci in D. mawsoni is the only known of Nototheniidae (Ozouf-Costaz et al. 1999; Pisano and occurrence beside the iceWsh Pagetopsis macropterus Ozouf-Costaz 2003). Boulenger, 1907, a member of the most derived noto- In both Dissostichus species, the metacentric chro- thenioid family Channichthyidae (Mazzei et al. 2004). mosomes of pair 1 and the submetacentric elements of Given the predominance of a single rDNA locus. In 123 Polar Biol notothenioids, we deduce that the extra locus in chro- in the antecedent of chromosome 3 of D. mawsoni. mosome pair number 3 of D. mawsoni represents a These underwent expansion through tandem dupli- locus gain, and that of the divergent P. macropterus a cation (Redi et al. 2001) in situ, possibly favored by separate event. The extra rDNA locus in D. mawsoni their position close to the centromeric heterochro- extends along the entire p arms of chromosome pair 3 matin, giving rise to the additional recognizable (Fig. 3e–h) while that of P. macropterus occupies a rDNA site. Evidence of rDNA-related sequence small region of a pair of submetacentric chromosomes elements dispersed throughout eukaryotic (Mazzei et al. 2004), consistent with diVerent mecha- genomes has been documented (e.g., De Lucchini nisms of locus evolution. et al. 1997; Stupar et al. 2002 and reference herein; Martins 2006). It has also been suggested that the Evolution of dual rDNA loci in D. mawsoni spread of such elements may occur frequently during evolution, with most events remaining unde- How did the additional rDNA chromosomal locus of tected due to the divergence of the sequences D. mawsoni arise? A survey of the organizational vari- (Falquet et al. 1997). ations of rDNA genes and proposed mechanisms may 2. Translocation—A second and more parsimonious provide some insight. Both quantitative and qualitative hypothesis is that a cluster of ribosomal genes variations of the ribosomal gene clusters occur could have been transferred from the original site between species as well as between individuals of the of chromosome number 2 to the acrocentric prede- same species. Qualitative heterogeneity is mainly due cessor of chromosomal pair number 3 through a to diVerences in the intergenic spacer regions (Long translocation, since chromosomal translocation is a and Dawid 1980). Quantitative heterogeneity consists frequent structural rearrangement in Wsh karyo- of variations in the extent of redundancy of rDNA cop- types (White 1973; King 1993). Gene ampliWcation ies, and in the number of ribosomal loci per genome and quantitative variations of heterochromatin (e.g., De Lucchini et al. 1997; Reed and Phillips 2000; could have preceded or followed such a transloca- Pedrosa-Harand et al. 2006). InterspeciWc and intraspe- tion, contributing to the increase in the size of the ciWc variations in the number of rDNA loci have been rDNA bearing chromosomal regions. described in several Wsh taxa (Zhuo et al. 1995; Rabova 3. Interchromosomal recombination—As a third pos- et al. 2001; Almeida-Toledo et al. 2002; Boron et al. sibility, we hypothesize that the presence of com- 2006 among others). A number of mechanisms have mon repetitive peri-centromeric sequences could been proposed to explain the changes in number and/ have enabled recombination to take place between or position of the rDNA chromosomal loci including the DNA region containing the rDNA site on chro- translocations (Hayasaki et al. 2001), dispersion of mosomes 2 and the centromeric region of the ante- rDNA repeats (Dubcovsky and Dvorak 1995; Schubert cedent of chromosome 3, leading to a new rDNA 1984; Raskina et al. 2004) and ectopic recombination locus on the latter. As in the previous hypothesis, (Pedrosa-Harand et al. 2006). gene ampliWcation and quantitative variations of With regard to the two Dissostichus species, the the associated heterochromatin could have con- morphological features of the two speciWc karyotypes tributed to further increase of the size of the rDNA suggest that the bi-armed chromosomes of pair 3 of D. bearing chromosome regions. mawsoni could have originated from a uni-armed ele- As an additional speculation, we cannot exclude that ment similar to one of the medium-sized acrocentric transposable elements could have been involved in the chromosomes in the karyotype of D. eleginoides. How mechanisms leading to changes of rDNA loci in Diss- this could have been accomplished would require ostichus since several classes of transposable elements detailed molecular analysis of the entire ribosomal are abundant in Wshes (Kidwell and Lisch 2000; VolV locus. However, based on karyotypic evidence and 2005) and possible relationships between transposable general mechanisms of expansion of repeated genes or elements and rDNA have been recognized (e.g., Burke sequences, we suggest at least three possible mecha- et al. 2003; Drouin 2000). In notothenioid Wshes, Tc1- nisms that could have led to the present double-rDNA like DNA transposon sequence and two families of chromosomal location in D. mawsoni. non-long terminal repeat retrotransposons, Rex 1 and 1. In situ ampliWcation of dispersed rDNA repeats— Rex 3 have been found (Capriglione et al. 2000; Ozouf- A Wrst hypothesis supposes that in addition to the Costaz et al. 2004), although no evidence of preferen- original rDNA locus on chromosome pair 2, a few tial accumulation of these transposable elements near dispersed repeats of ribosomal DNA were present the rDNA chromosomal sites has been conWrmed.

123 Polar Biol

Functional signiWcance of the dual rDNA loci possible applications in toothWsh stock assessment and in D. mawsoni Wshery management (Smith et al. 2001). In an evolutionary context, the data provided here Considering the very low frequency of dual rDNA loci can be a reference point for more extensive genomic among Antarctic notothenioids and the close phyloge- studies of cold-adapted notothenioids. Genome map- netic relationship between the two toothWsh, the dis- ping projects are completed or in progress for a num- tinct genomic diVerence in their rDNA loci was ber of Wsh species (reviewed in Phillips 2006), utilizing unexpected. Since the two species presumably large DNA insert clones that can be used for chromo- diverged about 14 million years ago (Near 2004), the some mapping by FISH, so we can expect that the diVerence in rDNA loci may be associated with the number of Wsh genomic studies will increase in the near evolutionary history of the two species in diVerent future, including for Antarctic species. Such genomic thermal environments. Does the extra set of ribosomal studies should be approached by integrating sequence genes in D. mawsoni serve a functional role in the analysis and chromosomal analysis, according to cytog- freezing habitats of the Southern Ocean? Although the enomic methodology (Jaillon et al. 2004; VolV 2005; present structural analysis cannot provide an answer to Pisano et al. 2006). Because D. mawsoni is a basal cold- this question, the dual rDNA loci in D. mawsoni quite adapted species among notothenioids, and genomic clearly reXects a genetic gain (rather than a loss in D. scale analyses of this species is currently in progress eleginoides), conceivably brought on by natural selec- (Cheng and Detrich 2006), the Antarctic toothWsh tion. It is reasonable to suggest that the doubling of could be a promising species for a cytogenomic charac- rDNA loci may increase ribosome production needed terization. for extra protein (antifreeze proteins) synthesis in the toothWsh living in freezing habitats, thus contributing Acknowledgments The Italian National Programme for Ant- to its survival. However, other Antarctic notothenioids arctic Research (PNRA), the Muséum national d’Histoire Natu- relle of Paris and IPEV (ICOTA Project), the US NSF OYce of (except for P. macropterus), living in similar cold envi- Polar Programs (OPP 0231006 to C.-H.C.C.) provided Wnancial ronments and possessing similar high levels of anti- support. The Australian Antarctic Division supported the partic- freeze proteins, do not display dual rDNA ipation of E. Pisano in the 1993 cruise on board the RSV Aurora chromosomal loci. We cannot exclude that quantitative Australis (Anare project N.728). We are grateful to the crews of R/V Nathaniel Palmer and R/V Tangaroa for logistic support increase of ribosomal genes did occur in other Antarc- during the ICEFISH 2004 Cruise (http://www.iceWsh.neu.edu) tic notothenioids in their evolutionary past, but was and Western Ross Sea Voyage 2004. The study is in the frame- perhaps not maintained, considering that no other work of the SCAR ScientiWc Research Program EBA (Evolution Antarctic notothenioid species reaches the large body and Biodiversity in Antarctica). size of D. mawsoni. Low temperatures depress the rates of biochemical and physiological reactions References (Hochachka and Somero 2002), and therefore the additional ribosome production by virtue of the extra Almeida-Toledo LF, Ozouf-Costaz C, Foresti F, Bonillo C, Por- rRNA genes may be a cold-adaptive compensatory to-Foresti F, Daniel-Silva MFZ (2002) Conservation of the mechanism for growth and development of this large 5S-bearing chromosome pair and co-localization with major rDNA clusters in Wve species of Astyanax (Pisces, Characi- species in freezing seawater. Consistent with this dae). 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